Agonists of A2A adenosine receptors (A2AARS) produce anti-inflammatory effects in bone-marrow-derived cells including neutrophils, monocytes, macrophages and T-cells. There is also evidence of functionally important A2AARs on cardiomyocytes and endothelial cells. We have shown that intravenous or subcutaneous injection into mice, rats, rabbits or dogs of very low doses of A2A agonists produces a profound inhibition of tissue inflammation and damage following ischemia/reperfusion, transplantation or infection. The overall goal of the studies proposed here is to understand the mechanism(s) of this tissue protection. Aim 1 is to study A2AAR interactions with ligands and G proteins at the molecular level by (A) radioligand binding studies to all four AR subtypes from multiple Species; (B) characterizing ligand-receptor interactions by rational design of new compounds aided by comformational field analysis (CoMFA) and by modifying receptors by site-directed mutagenesis; and (C) investigating ligand-receptor-G protein interactions by co-expression, purification and reconstitution strategies that utilize epitope-tagged receptors. Aim 2 is to use genetic approaches to (A) produce from congenic A2AAR KO mice, chimeric mice selectively lacking or expressing A2AAR5 in bone marrow-derived cells; and (B) use the Cre/IoxP strategy to create congenic lines of mice with the A2AAR gene (Aodra2a) selectively deleted in myeloid cells, monocytes/macrophages, endothelial cells and possibly other cells. Aim 3 is to examine cellular and signaling responses in inflammatory cell populations. Aim 3A is to investigate cellular and signaling responses in human neutrophils and monocytes. Aim 3B is to genotype and phenotypically characterize A2AAR responses in neutrophils, macrophages and primary endothelial cells harvested from wildtype, KO, chimeric and Cre/IoxP mouse lines. Murine macrophages and neutrophils will be purified from peritoneum or bone marrow and highly purified. Endothelial cells will be prepared by magnetic immunoaffinity chromatography from enzymatically dispersed lungs. These studies will be valuable because they will provide us with new information about how inflammatory cells contribute to tissue injury processes. We also will learn more about how A2AAR activation produces marked protection from infection and ischemia/reperfusion-induced injury. The use of A2AAR agonists and other compounds that inhibit inflammatory processes has great potential for treating ischemic and inflammatory diseases.